Molecular Mechanisms Regulating Cytokine Production by Human Neutrophils

Neutrophils are known to perform a series of effector functions that are crucial for the innate and adaptive responses towards infections. Furthermore, neutrophils respond to various stimuli, including microbial components, by synthetizing and secreting a variety of cytokines. In this context, the main objective of this study was to re-evaluate the capacity of human neutrophils to express and produce cytokines of the IL-17 family, including IL-17A, IL-17B, IL-17F and IL-17AF since the current literature on this topic is discordant. By performing RT-qPCR, immunohistochemistry (IHC), immunoblotting, protein measurement via commercial ELISA, chromatin immunoprecipitation (ChIP) and ChIP-seq, we evaluated transcriptional and epigenetic regulation, as well as production of the latter cytokines by highly pure (> 99.7 %) populations of human neutrophils. In agreement with some published data, we found that neutrophils do not express/produce IL-17A, IL-17F, IL-17AF or IL-17B mRNA/protein upon incubation with a variety of agonists. Similar findings were observed by analyzing neutrophils obtained from active psoriatic patients. No IL-17A and IL-17F mRNA expression/production was found even when human neutrophils from healthy donors were incubated with IL-6 plus IL-23 at very elevated concentrations in combination with inactivated hyphae or conidia from Aspergillus fumigatus, unlike shown in multiple studies. Moreover, consistent with the inability of human neutrophils to express IL-17A and IL-17F mRNA, no deposition of H3K27Ac and H3K4me1, which are histone marks of, respectively, active and poised genomic regulatory elements, was detected at the IL-17A/F genomic locus in resting or IL-6 plus IL-23-stimulated neutrophils. In addition, although we found that anti-IL-17A and anti-IL-17B commercial antibodies positively stained cytospin preparations of resting and activated neutrophils by IHC, these antibodies do not recognize any intracellular protein having the correct MW of either IL-17A or IL-17B in corresponding lysates of the same neutrophil preparations by immunoblotting. Since the same antibodies were found to strongly stain other intracellular proteins of neutrophils, we conclude that their ability to positively stain neutrophils derives from IL-17A- or IL-17B-independent unspecific binding. In conclusion, our data not only confirm and further support our previous original findings on the inability of human neutrophils to express/produce IL-17A, IL-17B and IL-17F mRNAs/proteins, but also attempt to explain why other published studies continue to report the opposite.